Lysine demethylases (KDMs) catalyze the removal of methyl groups from the side-chain amino groups of tri-, di- and mono-methylysines (Kme3/2/1), notably at specific sites in the N-terminal 'tails' of histones. There are two groups of KDMs: 1) the lysine-specific demethylases (LSDs 1 and 2), flavin dependent amine oxidases that can demethylate Kme2/1 and 2) the JmjC-domain-containing oxidases, Fe2+ and ?-ketoglutarate requiring enzymes that, in many cases, can also demethylate Kme3. Histone lysine methylations are epigenetic regulatory modifications that profoundly affect gene expression and aberrant histone lysine methylation occurs in a number of diseases including cancer. Excess activity of a number of particular KDMs is implicated in a variety of cancers. Interest in the discovery of drugs that inhibit KDMs is therefore growing but, especially for the JmjC enzymes, which is the largest of the two groups of KDMs. A factor which is slowing progress toward eventual development of drugs that target the JmjC KDMs is a lack of chemical probes, with probes defined as highly potent and highly specific inhibitors of individual JmjC KDMs. One circumstance that may be contributing to the lack of specific probes is that the existing inhibitor exclusively target the highly conserved binding site of the co-substrate, 2-oxoglutarate (2-OG) and/or chelate the active site Fe2+. In order to provide tools that may help remedy this situation, we are proposing to produce a comprehensive collection of JmjC KDM proteins, which will include full-length proteins, catalytic domain (demethylase) constructs and separate individual constructs for each of the many 'reader' domains (e.g. PHD or double Tudor domains) that form part of most, but not all, full-length JmjC KDMs. Once these proteins are in hand, we will develop at least one assay (binding or demethylation) and begin by screening all of them against a collection of FDA-approved drugs and compounds from clinical trials. The chemical profiles of the demethylase and reader domains thus generated will be incorporated in a database and made publicly available as a general aid to research in this area. The proteins that are produced in the process will become available as products and the assays that are developed will become services that we offer. Finally, these proteins will be screened against a much larger chemically diverse library (>80,000 compounds). Inhibitors that are identified will also become products. In Phase II we envision that some of these inhibitors will be used as the starting point for chemistry to produce more potent probes or to produce fluorophor-labeled probes for fluorescence polarization assays.